The PPP2R2A gene encodes B55?, a B regulatory subunit of PP2A, which is found hemizygously deleted at high frequency in prostate, ovarian and luminal B breast cancer. PP2A is a Ser/Thr phosphatase that consists of a collection of trimeric holoenzymes whose substrate specificity, and thus biological function, is determined by the regulatory B subunit. There are at least fourteen known genes encoding different B subunits belonging to four unrelated gene families. While it is well known that PP2A plays a key tumor suppressor function, which inhibition is required for transformation of a variety of human normal epithelial cells and fibroblasts in cooperation with defined sets of oncogenes and inactivated tumor suppressor genes, the actual holoenzymes implicated are not well understood. Data from TCGA and others shows that PPP2R2A is deleted in the majority of prostate tumors (>50% hemizygous/~7% homozygous loss). Importantly, PPP2R2A hemizygous loss correlates with its reduced mRNA expression. Moreover, PCa patients present reduced disease free survival with this alteration. While this has led to the suggestion that PPP2R2A is a tumor suppressor, there is not data supporting that loss of this gene contributes to PCa. We have found that limited ectopic expression of B55? in PCa cell lines, which naturally express lower levels than normal prostate epithelial cells and other PCa cell lines, results in dramatic toxicity. This toxicity is linked to mitotic arrest, euploidy, cell death, and potent tumor growth inhibition in SCID mice. Our preliminary studies also show growth inhibition and architecture defects in PCa and DU145 3D organoids following inducible upregulation of B55?. Moreover, treatment of PC3 cells with phenothiazines, which bind and activate PP2A, mimic cell cycle effects seen with B55? reconstitution. We hypothesize that PPP2R2A hemizygous deletion and/or other alterations that reduce B55? expression in PrECs promote transformation and tumorigenicity and restoring B55?/PP2A activity has therapeutic potential. To test this hypothesis we propose two aims: (1). To determine the transforming potential and tumorigenicity of B55? loss/downregulation in Prostate Epithelial Cells (PrECs) and PCa cell lines and the effect of reconstitution of B55? expression in their ability to differentiate, grow and/or invade when grown as 3D organoids. (2). To determine if the growth suppressor function of B55? in 3D organoids is associated with its mitotic functions and/or the result of alterations in survival/differentiation pathways and if it can be targeted with PP2A activating drugs. Successful completion of this work will support the feasibility of multiple studies aimed at understanding (a) oncogene/tumor suppressor gene cooperativity in prostate tumor development in mice, and its correlation in human tumors; (b) signaling mechanisms and identification of key target substrates; and importantly, (c) devising strategies to therapeutically upregulate this holoenzyme in tumor cells. Because PPP2R2A alterations occur at high frequency in prostate tumors, the proposed studies will lay the molecular and biological framework for the development of novel classes of PP2A drugs that could potentially help treat the large pool of PCa patients with hemizygous deletions on PPP2R2A.